Electron transfer between protein partners (e.g., substrates, products, other proteins, and small effector molecules) is a key component of many important biological processes. Kinetic and thermodynamic studies suggest that the electron transfer of many enzymes (and thus their overall reaction mechanisms) is regulated by the binding of protein partners. The acyl-CoA dehydrogenases (ACDs) that play a major role in b-oxidation, and ribonucleotide reductase (RNR), which provides deoxynucleotides for DNA synthesis, are both involved in electron transfers that are highly regulated by protein partner binding. The redox properties of their noncovalently-bound protein cofactors (flavins and diiron centers, respectively) have been found to be excellent indicators of the thermodynamic changes linked to regulation, since substrate/product binding perturbs the midpoint potentials of these reporter groups. Thus, spectroelectrochemistry can often identify changes that cannot be observed by any other means. The objective of this proposal is to gain additional insight into the interactions involved in protein partner binding that result in the catalytic regulation of the ACDs and RNR. For the ACDs, our goal is to learn how the interactions induced by ligand binding affect catalysis and the thermodynamic properties of both the ACD and the ligand. A wealth of thermodynamic data show that the ACDs are highly regulated. Recent Raman spectroscopic and redox studies using the same Raman active product analog have led us to hypothesize that substrate binding causes the redox potential of the enzyme (E) to change, and that in turn, binding to the enzyme causes the substrate (S) to be polarized in the active site, demonstrating that the properties of the activated complex [E.S] are different from those of the free E and S. In order to learn more about the activated complex [E.S], we have devised a new series of substrate analogs to probe that species, further exploring substrate polarization and changes in redox chemistry. For RNR, we propose to probe the interactions responsible for the gating of the long-range electron transfer between subunits. Redox potential studies of the iron center have provided the only data indicating that R1 binding affects R2. The R2 iron center acts as a reporter group for conformational changes and/or long- range interactions that result from the formation of the [R1.R2] complex, and we expect to observe additional changes caused by the binding of substrate, product, and allosteric effectors.